Modulating protein-protein interactions represents an attractive goal both for probing protein function and for therapeutic applications. However, developing modulating agents using traditional small molecule-based approaches is quite challenging. This is due to the large and irregularly shaped interfaces inherent in protein-protein interactions. Because of their relatively large size and ability to mimic natural protein surfaces, synthetic peptides and proteins can be developed to bind target protein surfaces with high affinity and selectivity. However, peptides and proteins suffer from several significant disadvantages, in particular low bioavailability due to their rapid degradation by proteases. As a general proposition, proteolytic degradation of peptide pharmaceuticals limits the practical scope of their therapeutic use.
Various groups have sought to target large and complex protein-protein interaction interfaces using a variety of different classes of compounds, including peptides derived from phage display, antibody conjugates, nucleic acid aptamers, and rigid macrocyclic peptide scaffolds. In one recent example, mirror-image phage display was used to develop a D-peptide targeting vascular endothelial growth factor (VEGF). Although this approach yielded a 56-amino acid D-peptide that is anticipated to have decreased susceptibility to proteolytic degradation, it required the total chemical synthesis of the D-VEGF protein. This limits its general utility for developing inhibitors of other protein targets because it is not cost-effective (and for larger proteins, not possible) to fabricate the D-stereoisomer of a natural L-protein target.
It has been shown that peptide foldamers in which a subset of the residues contain backbone modifications in the form of β-amino acid residues α/β-peptides) can effectively mimic α-helices in the disruption of helix-mediated protein-protein interactions. Such oligomers can show high affinity and selectivity for target proteins and are less susceptible to proteolytic degradation than α-peptides composed exclusively of natural α-amino acid residues. Recently, it has also been shown that α/β-peptides can also have improved pharmacokinetic properties in vivo over their α-peptide counterparts, validating compounds of this type for potential therapeutic applications. See U.S. Pat. Publ. 2013/0177981, published Jul. 11, 2013.
Vascular endothelial growth factor (VEGF) is a soluble homodimeric protein which binds two cell-surface receptor tyrosine kinases, VEGFR1 and VEGFR2, to trigger receptor dimerization, phosphorylation, and intracellular signaling to initiate angiogenesis. Because of its critical role in angiogenesis, antagonists of the VEGF/VEGFR interaction are currently used in the treatment of both cancer and wet macular degeneration. The receptor recognition site on VEGF is large and topologically complex, with over 800 Å2 of the surface of VEGF buried upon receptor binding. The receptor recognition site appears to be representative of many protein-protein interactions that interact primarily though flat, hydrophobic surfaces. α/β-Peptide analogs of phage-derived peptide v114 that bound VEGF with modest binding affinity (Ki=˜2-5 μM in our fluorescence polarization (FP) assay), inhibited VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) in culture, and had a reduced susceptibility to proteolytic degradation relative to the parent α-peptide have been reported in the literature. Haase, H. S.; Peterson-Kaufman, K. J.; Lan Levengood, S. K.; Checco, J. W.; Murphy, W. L.; Gellman, S. H. “Extending Foldamer Design beyond α-Helix Mimicry: α/β-Peptide Inhibitors of Vascular Endothelial Growth Factor Signaling. J Am Chem Soc. 2012, 134, 7652-5. However, because of v114's irregular conformation when bound to VEGF, the strategies employed for mimicry of this peptide are not general for mimicry of other protein-protein interaction inhibitors. Thus, there is a long-felt and unmet need for a method that can be widely applied to generate α/β-peptides to modulate a variety of different proteins of interest starting from peptides that bind to these proteins.
Protein-based affinity reagents derived from well-defined, non-immunoglobulin scaffolds offer an alternative to antibodies for selective and high affinity recognition of proteins in modulating protein-protein interactions, protein targeting, and imaging. The “Z-domain” scaffold derived from the domain B of staphylococcal protein A, is a relatively stable, three-helix bundle protein that presents a large “protein binding face” on helices 1 and 2. The protein binding face can be altered to selectively bind desired proteins using combinatorial approaches. The wild type Z-domain (Z-IgG, FIG. 1A) binds the Fc portion of IgGs. Randomization of up to 13 protein-contacting residues in helices 1 and 2 can be used to develop Z-domain peptides that bind a variety of different proteins. For example, a peptide adopting the Z-domain structure has been developed to bind HER2 (Z-HER2), a cell surface receptor overexpressed on many cancer cells. The Z-HER2 compound is useful for tumor targeting and diagnostic imaging and recently reported clinical data has validated its use for the imaging of breast cancer tumors in humans. Peptides derived from the Z-domain scaffold have also been developed to antagonize the binding of several soluble proteins such as VEGF and tumor necrosis factor-α (TNFα) to their cognate receptors (Z-VEGF and Z-TNFα, respectively, FIG. 1A). These compounds provide potential alternatives to antibodies for the selective therapeutic inhibition of their respective interactions or for analyte detection/capture. In 1998, the biotechnology company Affibody Biotechnology AB (Bromma, Sweden) was founded to develop Z-domain-based peptides for use as antibody alternatives. (“AFFIBODY” is a registered trademark in the United States for use in association with pharmaceutical preparations for the treatment of cancer and infectious diseases, and for diagnostic preparations or reagents for clinical and medical laboratory use.)
Two-helix analogs of the Z-IgG and Z-HER2 have been reported that stabilize the intended conformation with several α-amino acid substitutions and an interhelical disulfide bond. In the case of the two-helix Z-HER2 analog, the incorporation of helix-promoting 2-aminoisobutyric acid (Aib) residues throughout the sequence (which may be used to increase resistance to proteolytic degradation) lead to an 8000-fold decrease in binding affinity for HER2 relative to the full length Z-HER2. Incorporation of only two of these Aib substitutions lead to a high binding two-helix peptide, though this still bound HER2 with affinity weaker than the parent Z-domain. See, for example, U.S. Pat. No. 8,198,043, issued Jun. 12, 2012.